Promoter and Use Thereof
a promoter and promoter technology, applied in the field of bioengineering technology, can solve the problems of non-rational work, time-consuming and tiring screen work, and not being able to screen out excellent transformants, etc., and achieves the effect of not being able to achieve excellent transformants, useful for scientific purposes, and being uneconomical for industrial purposes
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embodiment 1
[0026]Genome Extraction from A. niger
[0027]Conidia of A. niger (1×106) were inoculated in 100 mL malt extract liquid medium (3% malt extract and 0.5% tryptone) at 35° C. and 250 r / min for 48 h. The mycelia were harvested with Miracloth (Calbiochem, San Diego, Calif., USA), washed with sterile water and frozen in liquid nitrogen. Tissues were ground by Liquid nitrogen grinding, and the genome DNA of A. niger was isolated with a DNeasy Plant Mini Kit (QIAGEN, Germantown, Md., USA).
embodiment 2
Obtaining Promoters Induced in Acidic Conditions (by Low pH)
[0028]A. niger gene expression data (Accession, GSE11725) in NCBI GEO Datasets were analyzed to detect changes in mRNA levels from pH 4.5 to pH 2.5 and 4 genes was identified for increased gene expression with decreased pH value. The sequence 1500 bp upstream of the start codon ATG was analyzed using Neural Network Promoter Prediction software (version 2.2) (http: / / www.fruitfly.org / seq_tools / promoter.html) and all can be identified with a transcription start site, and the predicted promoters were named as Pgas, PpatI, Ppth, and Paat.
[0029]Pgas was amplified from the A. niger genome using the primers gas-F (SEQ ID NO.2) and gas-R (SEQ ID NO.3) with restriction sites Eco RI and Sma I at the 5′ and 3′ ends, respectively. PpatI was amplified from the A. niger genome using the primers pat-F (SEQ ID NO.4) and pat-R (SEQ ID NO.5) with restriction sites Sac I and Bam HI at the 5′ and 3′ ends, respectively. Ppth was amplified from t...
embodiment 3
Construction of Expression Cassette of Promoters Induced in Acidic Conditions
[0031]GFP (SEQ ID NO.10) was synthesized with coden optimization and contained Bam HI and Pst I restriction sites at the 5′ and 3′ ends, respectively. Trp terminator (Ttrp) was PCR amplified with primers Ttrp-F (SEQ ID NO.11) and Ttrp-R (SEQ ID NO.12) using pAN7-1 as a template, and restriction sites Pst I and Hin dIII was added to the 5′ and 3′ ends, respectively. Ttrp was digested with Hin dIII and Pst I, GFP was digested with Bam HI and Pst I, and the two sequence were ligated to pUC18 digested with the same enzyme, and pGT was obtained. GFP-Ttrp was amplified with the primers GFP-F1 (SEQ ID NO.13) and Ttrp-R using pGT as a template and reversely connected to pMD19-T vector (Takara, Tokyo, Japan) to generate pMD-GFP-Ttrp. Pgas and pMD-GFP-Ttrp were digested with Eco RI and Sma I and connected to generate the Pgas-GFP-Ttrp expression vector. For co-transformation, the Pgas-GFP-Ttrp expression cassette was...
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